Enzymes are central components of nearly all metabolic and signaling pathways in cells and tissues. The dysregulation of enzymes and their endogenous inhibitory proteins contributes to the development of several diseases, including cancer. Nonetheless, efforts to elucidate the function of specific enzymes in cancer have, to date, suffered from a lack of techniques that can assess the activity of these proteins in complex biological systems. To address this problem, we have introduced a chemical proteomic strategy termed activity-based protein profiling (ABPP) that utilizes active site-directed probes to measure changes in enzyme activity directly in native proteomes. To date, we have generated activity-based probes that target numerous enzyme classes, including proteases, lipases, histone deacetylases, and cytochrome P450s. In the previous funding period, we applied ABPP to identify several enzyme activities dysregulated in aggressive human cancer cells, including the serine proteases uPA and tPA, and the previously uncharacterized transmembrane hydrolase KIAA1363. We have shown that these enzymes play important roles in supporting the malignant properties of cancer cells, although the biochemical mechanisms for these effects remain to be fully elucidated. We have also introduced advanced tag-free versions of ABPP that exploit the versatility of click chemistry and resolving power of mass spectrometry to enable high-content profiling of small molecule-protein interactions in living systems. Finally, we have developed a complementary proteomic platform for globally mapping the endogenous substrates of proteases termed PROTOMAP (PROtein TOpography and Migration Analysis Platform). In this competitive renewal application, we will apply our suite of ABPP and PROTOMAP technologies to test three major hypotheses of high significance to the fields of cancer and chemical biology: 1) KIAA1363 promotes cancer aggressiveness through regulation of an ether lipid signaling network, 2) dysregulated proteases contribute to cancer pathogenicity by activating and/or inactivating key signaling pathways, and 3) tag-free ABPP will offer a general and quantitative technology to map small molecule-protein interactions in living systems. We anticipate that these studies will define key enzymatic pathways that support cancer malignancy and contain new biomarkers and therapeutic targets, as well as produce methodological advances that greatly expand the scope and utility of the ABPP and PROTOMAP technologies.